PROJECT SUMMARY/ABSTRACT Objective. Gene therapy has the potential to permanently correct or prevent monogenic disorders such as cystic fibrosis. We have a demonstrated track record of utilizing many categories of viral and non-viral vectors to deliver genes to the airways; however, choosing the best gene therapy vector leads to a very significant dilemma. Non-integrating vectors may not persist and integrating vectors may cause insertional mutagenesis. There is a critical need for improved gene delivery tools that address this conundrum. Our long-term goal is to engineer a vehicle for gene delivery to cells that is a safe and effective therapeutic for cystic fibrosis. To this end, the goal for these proposed studies is to develop an integrating vector with a predictable integration pattern that targets 'safe harbor' genomic loci. Nonviral vector systems are used increasingly as tools for gene transfer applications. We successfully used the piggyBac DNA transposon system as an effective integrating vector for gene transfer. Method. Based on our preliminary studies, piggyBac transposase is amenable to modification. Furthermore, recent advances in the ability to engineer customized zinc finger proteins make the possibility of targeted transposition promising as a therapeutic approach. The overall hypothesis is that the piggyBac transposon system may be modified to retarget integration. We will use multiple approaches to direct the piggyBac transposase to designated loci, increase vector delivery efficiency, and improve the utility and safety of the vector for gene therapy. Here we propose to: 1) generate and validate the function of 'safe harbor' zinc finger protein/piggyBac transposase fusion proteins; 2) map zinc finger protein/piggyBac transposase mediated transposon integrations in the genome; and 3) create a hybrid piggyBac/lentivirus vector system to improve delivery efficacy. These studies will provide important mechanistic information regarding the motifs important for directing piggyBac integrations.